• Oxidation of Dietary Amino Acids Disrupts their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells

      El Refaey, Mona M.; Department of Cellular Biology and Anatomy (2016-07)
      Age-dependent bone loss has been well documented in both human and animal models. Since it has been proposed that aging is associated with an increase in the generation of damaging reactive oxygen species (ROS), our hypothesis was that the oxidized products of dietary amino acids could play a role in age-induced bone loss by altering osteoprogenitor cell differentiation and function or activating osteoclastic activity. We first examined the effects of the oxidized nutrients on the bone marrow-derived mesenchymal stem cells and our data showed a decrease in the protein and gene expression of osteogenic markers normally stimulated by nutrients. Aromatic amino acids activated signaling pathways involved in protein synthesis in vitro, and thus, in contrast, the oxidized metabolites of these aromatic amino acids had no effect on the activation of these anabolic pathways. We then examined the bone marrow concentration of the oxidized aromatic amino acids in mature (12 months) vs. aged (24 months) C57BL/6 mice and found that kynurenine, the oxidized product of the aromatic amino acid tryptophan, was found in the highest concentration in 12 months mice. Thus, we tested the effects of kynurenine, fed as a dietary supplement, on the bone mass of twelve-month-old C57BL/6 mice compared to a normal protein diet to see if the oxidized amino acid would induce a pattern consistent with age-related bone loss. Twelve-month-old, male C57BL/6 mice were fed one of four diets; 18% protein diet (normal protein diet); 8% protein diet + tryptophan; 8% protein diet + kynurenine (50 μM) and 8% protein diet + kynurenine (100 μM) for 8 wks. Bone densitometry and micro-CT analyses demonstrated bone loss following the kynurenine diet. Histological and histomorphometric studies showed a decreased bone formation and an increased MONA M. EL REFAEY Oxidation of Dietary Amino Acids Disrupts Their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells (Under the direction of DR. CARLOS M. ISALES) osteoclastic activity in the kynurenine groups; these animals also exhibited an increase in serum pyridinoline, a marker of bone breakdown. Thus, these data demonstrate that feeding an oxidized product of an essential amino acid induces bone loss in a pattern consistent with accelerated aging, and we propose that one of the mechanisms involved in age-induced bone loss may be from alterations of dietary nutrients by the increased generation of ROS associated with aging.
    • Signaling Mechanism of Blood-Retinal Barrier Regulation: Role of Mitogen-Activated

      Yang, Jinling; Department of Cellular Biology and Anatomy (2011-03)
      Breakdown of the blood-retinal barrier (BRB) is an early hallmark of diabetic retinopathy. A critical component in retinal vascular hyper-permeability is increased production of vascular endothelial growth factor (VEGF). VEGF is a potent permeability factor that activates mitogen-activated protein (MAP) kinases. Pigment epithelium-derived factor (PEDF), an endogenous anti-permeability factor, blocks VEGF-induced vascular permeability increase. However, the mechanisms underlying the actions of VEGF and PEDF in regulating endothelial permeability are not yet clear. Previous studies in our laboratory have shown that VEGF induces paracellular permeability via beta-catenin nuclear translocation/transcriptional activation and subsequent upregulation of urokinase plasminogen activator receptor (uPAR). This current study tests the role of two MAP kinases, p38 and extracellular-signal regulated kinase (ERK), in regulating VEGFinduced beta-catenin signaling, uPAR expression and BRB breakdown. We also evaluate the effects of PEDF on this VEGF permeability inducing pathway. The role of MAP kinase in this VEGF permeability inducing pathway was first evaluated using inhibitors of p38 and ERK. These inhibitors preserve the endothelial barrier function upon VEGF treatment. In confluent endothelial cells, cytosolic beta-catenin is phosphorylated by glycogen synthase kinase (GSK) then ubiquitinated and degraded. With VEGF treatment, GSK is phosphorylated/inactive followed by beta-catenin cytosolic accumulation, nuclear translocation and subsequent uPAR expression. These effects were blocked by MAP kinases inhibitors. This indicates p38 and ERK as mediators of VEGF-induced beta-catenin signaling, uPAR expression and endothelial barrier breakdown. Next, it was found that PEDF not only blocks VEGF-induced endothelial permeability increase and MAP kinase activation but also prevents the activation of GSK/beta-catenin signaling as well as uPAR expression. However, PEDF did not block VEGF receptor-2 (VEGFR-2) phosphorylation suggesting that PEDF acts downstream of VEGFR-2 and upstream of MAP kinase level. To further evaluate the role of p38 in regulating VEGF-induced permeability, adenovirusmediated delivery of p38alpha mutants was used. One p38alpha mutant has an altered ATP-binding site thus looses its activity. It is more efficient in blocking VEGF-induced GSK/beta-catenin signaling, uPAR expression and paracellular permeability increase. This study identifies p38alpha and ERK as mediators of VEGF permeability-inducing signaling. They could also serve as potential therapeutic targets for diseases featured by blood-retinal barrier dysfunction.
    • The role of stromal cell-derived factor-1 in cell mobilization, cell homing and neovascularization following stroke

      Walker, Aisha L.; Department of Cellular Biology and Anatomy (2007-11)
      Stroke is the 3rd leading cause of death and the leading cause of long-term disability in the U.S. With only one approved drug presently used in clinics, there is a great need for the development for new therapeutic targets. Stromal cell derived factor-1 (SDF-1) is a small chemokine that may aid in cerebral repair following stroke. Acting primarily through the CXCR4 receptor, SDF-1 is known to be chemotactic for neuroblasts, endothelial cells, and bone marrow derived (BMD) cells including stem and progenitor cells found in the bone marrow. Recently, BMD stem/progenitor cells have become widely studied for their potential role in tissue repair following ischemia. SDF-1 is under hypoxic regulation and is highly expressed in ischemic brain tissue for at least 30 days following ischemia suggesting it may play role in long term repair or remodeling. The goal of these studies is to determine the role of SDF-1 in cerebral repair following stroke. I hypothesize that SDF-1 upregulaton during brain ischemia contributes to tissue repair and neurological recovery by inducing the homing of bone marrow-derived cells to the site of injury and neovascularization. In a mouse middle cerebral artery ligation (MCAL) permanent occlusion stroke model, I investigated mobilization, homing, and differentiation of adult bone marrow derived (BMD) cells in response to SDF-1 induced by cerebral ischemia. Results presented in this dissertation show that SDF-1 induces mobilization of BMD cells following stroke. Once mobilized, BMD cells homed to the brain and either retained their blood cell phenotypes (i.e. monocytes and neutrophils) or they differentiated mostly into microglia cells. Many BMD cells migrated to a perivascular location with a subset becoming pericytes. Additionally, I found that SDF-1 induced neovascularization and this occurs through a combination of angiogenic and vasculogenic processes in the in vivo stroke model as well as in an in vitro tube formation assay. However, we did not detect beneficial preservation of brain tissue or augmented functional recovery with treatment of SDF-1, but it remains to be determined if altering timing, delivery, or isoform-specificity of SDF-1 may be therapeutically beneficial.
    • Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • Mechanisms of Homocysteine-Induced Retinal Ganglion Cell Death

      Ganapathy, Preethi S.; Department of Cellular Biology and Anatomy (2010-12)
      The purpose of these studies was to determine the effect of excess homocysteine on retinal ganglion cell viability. An overview of homocysteine metabolism and the literature concerning homocysteine-induced neurotoxicity is given below, followed by detailed descriptions of the eye, the retina, and retinal ganglion cells.
    • PKC and ATR Mediated Regulation of Cisplatin-Induced Renal Tubular Cell Apoptosis

      Pabla, Navjotsingh; Department of Cellular Biology and Anatomy (2009-03)
      Cisplatin is one of the most widely used anti-cancer drug. However, its use and efficacy is limited due to nephrotoxicity. One fourth of patients treated with cisplatin develop varying degree of renal impairment, frequently resulting in acute kidney injury. Due to high mortality associated with acute kidney injury, effort has been made to understand the molecular basis of cisplatin nephrotoxicity and develop effective renoprotective strategies. In kidneys, cisplatin is accumulated in tubular cells; however the uptake mechanism that is responsible for high accumulation of cisplatin in renal cells is unclear. In tubular cell, cisplatin accumulation induces cell death by apoptosis. Mechanistically, our laboratory has demonstrated a critical role of p53 in tubular cell apoptosis during cisplatin nephrotoxicity. However, the proximal events that contribute to p53 activation and related signaling are unknown. The focus of my work was to decipher these early events during cisplatin nephrotoxicity. Firstly, my results suggest that the copper transporter Ctr1 is highly expressed in renal tubular cells and is responsible for renal uptake of cisplatin. Secondly, I show that DNA damage response involving ATR-Chk2 is responsible for p53 activation and consequent apoptosis during cisplatin-induced kidney injury and nephrotoxicity. Thirdly, I have identified that PKCd is a novel regulator of cisplatin nephrotoxicity. During cisplatin treatment PKCd is activated in a Src dependent manner and is responsible for activation of MAPKs, contributing to renal cell death. Most importantly, my results suggest that pharmacological inhibition of PKCd ameliorates renal injury without affecting the anticancer efficacy of cisplatin. These results have not only provided new insights into the 3 molecular mechanism of cisplatin nephrotoxicity, but have also identified a novel strategy to mitigate the side effects of cisplatin in normal renal tissues.
    • Murine CD19+ Plasmacytoid Dendritic Cells Expressing Indoleamine 2,3 Dioxygenase

      Kahler, David J.; Department of Cellular Biology and Anatomy (2008-10)
      Indoleamine 2,3 Dioxygenase (IDO) is a potent immunomodulatory enzyme whose role has been described in diverse physiologic states including pregnancy, cancer, tissue transplants, autoimmune disease, chronic inflammation, and depression. IDO suppresses antigen specific T cell proliferation via mechanisms including tryptophan degradation and the production of toxic metabolites, and the activation of resting regulatory T cells (Tregs). IDO expression is tightly regulated in the murine spleen, as only rare dendritic cell (DC) subsets are competent to express IDO. Therefore, an accurate phenotype by which to identify IDO competent DCs in tissues is important when ascribing the role of IDO competent DCs in disease models. Here we show that IDO competent CD19+ pDCs (CD19+ pDCs) express high levels of costimulatory receptors (CD80 / CD86) under homeostatic conditions indicating a mature or activated phenotype and uniquely express the Class I MHC-like molecule CD1d, and the chemokine receptor CCR6. IDO competent pDCs do not share the same lineage as other murine splenic DCs as they were the only DC subset to express Pax5, and were present in reduced numbers in murine models of B cell development indicating that they develop from B cell precursors. Distinct signaling requirements regulate IDO induction in IDO competent pDCs as MyD88 was required for IDO induction and function in inflamed skin draining lymph nodes following phorbol myristate acetate application but not for IDO transcript expression or STAT1 or STAT2 protein phosphorylation following treatment with recombinant cytokines. CD19+ pDCs from WT mice but not mice genetically deficient for the IDO1 gene formed stress granules (SG) following treatment with IFNγ, which were not prevented by inhibitors of IDO activity indicating that SG formation was not IDO dependent. We hypothesize that IDO competent murine splenic pDCs uniquely expressing CD19 are phenotypically and functionally distinct from other splenic DC subsets and respond to inflammatory signals by expressing IDO. We further hypothesize that activated IDO causes distinct yet undefined biochemical changes within IDO competent pDCs following induction most probably by activating the integrated stress response and the eif2a kinases GCN2, PKR, and PERK.
    • The Role of Stromal Cell-Derived Factor-1Β in Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells and Bone Formation

      Herberg, Samuel A.; Department of Cellular Biology and Anatomy (2013-03)
      The experiments performed for this dissertation tested the hypotheses that SDF-1β enhances osteogenic differentiation of BMSCs, promotes engraftment and bone formation following whole-body irradiation, and potentiates suboptimal BMP-2 osteoinduction in a model of acute bone injury. We used multipotent primary BMSCs from 18-month-old C57BL/6J mice, genetically modified to overexpress SDF-1β, to ask whether SDF-1β played a role in cell survival and osteogenic differentiation of BMSCs in vitro. Our studies revealed that SDF-1β protected BMSCs from oxidative stress through increasing autophagy and decreasing apoptosis, independent from potential effects on cell proliferation. In support of the hypothesis we also found that SDF-1β enhanced calcium mineral deposition (independent of BMP-2 co-stimulation), upregulated key osteogenic markers, and increased phosphorylation of intracellular Erk1/2 and Smad1/5/8, thereby potentiating BMP-2 signal transduction during osteogenic differentiation, which was attenuated by blocking CXCR4 signaling. We next inquired whether SDF-1β promotes BMSC engraftment and new bone formation. Using direct tibial transplantation in irradiation-preconditioned animals, we found that SDF-1β enhanced new trabecular bone formation upon local BMSC transplantation. The data furthermore suggested that the differential proteolytic clearance of SDF-1 splice variants in the systemic and local environment following myeloablative injury may be an important determinant in the success of stem cell therapy protocols. The suggestion that SDF-1β could regulate BMP-2 osteoinduction through regulating CXCR4 signaling was compelling because several studies have reported a comparable effect using SDF-1α. We examined the direct contribution of SDF-1β to BMP-2 osteoinduction in a critical-size calvaria osteotomy model and found a dose-dependent ability of SDF-1β to potentiate suboptimal BMP-2-induced bone formation to levels comparable to those obtained with the 10-fold higher optimal/benchmark BMP-2 dose, which was blunted by perturbing CXCR4 signaling. These in vitro and in vivo findings expand our understanding of BMP-2 osteoinduction and implicate osteogenesis-enhancing properties of SDF-1β pointing towards its translational potential for cell therapy and regenerative medicine applications. It appears feasible for SDF-1β to improve bone regeneration in a variety of orthopaedic situations and ultimately reduce the burden of musculoskeletal injuries.